Our work focuses on specialized synapses in the inner retina. We have expanded our study of inhibitory synaptic connections made by amacrine cells within the inner retina, to understand how feedforward and feedback inhibition contributes to signal processing in this network. We previously discovered that A17 amacrine cells provide rapid GABAergic feedback to rod bipolar cell ribbon synapses via a release process that is independent of membrane depolarization or voltage-gated calcium channels (Chavez, et al., 2006). This rapid feedback may be essential to prevent the rapid depletion of readily-releasable vesicles from the rod bipolar cell synaptic terminal (Singer and Diamond, 2006). Our most recent work indicates that reciprocal feedback from A17s extends the range over which these synapses encode luminance and compute contrast (Oesch and Diamond, submitted). Feedback from other amacrine cells weakly modulates the synaptic gain but does not change the operating range. We also find that A17-mediated feedback inhibition enhances the gain of synaptic responses in the rod pathway to the absorption of single photons (Grimes, et al., 2015). Our understanding of ribbon synaptic physiology in bipolar cells is limited to rod bipolar cells. We don't know as much about synaptic transmission from cone bipolar cells, because it is very difficult to obtain synaptically coupled cone bipolar - ganglion cell pairs. We have crossed mouse lines with genetically encoded markers identifying specific types of bipolar and ganglion cells that are very likely to be connected. In particular, we have utilized mouse lines in which type two cone bipolar cells (CBC2) can be visualized. We find that CBC2s make reciprocal synaptic connections with AII amacrine cells. We are studying bidirectional communication between these two cell types, and we are also studying how the rod bipolar cell signal is shaped by the AII amacrine cell before it is passed to the CBC2. Our preliminary findings suggest that AIIs preferentially transmit information about contrast, but not luminance, to CBCs. We are examining the cellular and synaptic processes that underlie this transformation. We plan to expand these experiments to examine release from ON CBCs and their postsynaptic partners in an effort to compare the dynamics of vesicle release from ON and OFF bipolar cells. We have also collaborated with Donald Zack (Johns Hopkins) to examine the physiological characteristics of retinal ganglion cells derived from stem cells. We find that these cells develop divers firing properties that are consistent with different ganglion cell subtypes at different stages of development. We also found that ESC-derived ganglion cells express functional glutamate receptors. A revised manuscript has been submitted for publication. Finally, we are extending our electron microscopy studies, in collaboration with Tom Reese and Richard Leapman, to explore the detailed ultrastructure of synaptic ribbons in photoreceptors and rod bipolar cells. So far, EM tomography enables us to detect protein filaments that tether synaptic vesicles to the ribbon and the presynaptic membrane. This approach may enable us to discern morphologically, for the first time, docked and primed synaptic vesicles. We have obtained detailed reconstructions of entire, intact ribbons (Graydon, et al., 2014) and are now working to obtain higher resolution images to examine quantitatively the tethers that connect synaptic vesicles to the ribbon and to the presynaptic membrane. To this end, we have recently obtained high-resolution images of ribbon synapses using high-pressure freezing EM techniques. This approach has been used to great effect in cultured mammalian neurons, but it has proved quite difficult in intact tissue. A great deal of effort has finally enabled us to obtain high-quality freezing the in the inner plexiform layer of intact retina, which will enable us to address numerous questions of synaptic function.